CREATINE-KINASE - THE REACTIVE CYSTEINE IS REQUIRED FOR SYNERGISM BUT IS NONESSENTIAL FOR CATALYSIS

Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mi(b)-CK) with either glycine, serine, alanine, asparagine, or aspartate, The resulting mutant Mi(b)-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mi(b)-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mi(b)-CK were significantly inhibited under the same conditions. At low chloride concentrations, the V(max) of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 11-fold increase in K(m)(PCr), but only 1.7- and 2.8-fold reductions in V(max), respectively, compared to wild-type Mi(b)-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (i.e., K(d) > K(m)). We confirmed this finding for wild-type Mi(b)-CK by determining the K(d) and K(m) values for both substrates in the forward reaction. Analysis of these constants for the two mutant enzymes C278G and C278S showed that the reactive cysteine (1) is not directly involved in binding either substrate (K(d) values for mutants were not dramatically changed compared to the wild type) and (2) is necessary for synergistic substrate binding (K(d) values for mutants were smaller than the corresponding K(m) values). These results suggest that the reactive cysteine is necessary to confer conformational changes upon substrate binding and support the proposal that this residue has a role in shaping the active site, possibly by acting as a hinge between the two substrate binding sites.